The translocation of proteins and RNA between the cytoplasm and nucleus is mediated by a family of soluble transport receptors, of which only nine have so far been identified in mammals. The importance of understanding these factors is underscored by findings that correlate the improper localization of key regulatory proteins to disease states such as carcinogenesis. In addition, several transporters play integral roles in the life cycle and pathogenesis of viruses (e.g., HIV). The proposed research plan is based on the identification and cloning of a novel, human transport receptor, karyopherin X. The objectives are to biochemically characterize karyopherin X, identify its cargo and map the motif it recognizes and binds to within the cargo sequence. To realize these goals, two specific aims will be pursued. Aim number 1 is to determine if this karyopherin shares the biochemical properties that define the members of the importin-beta-like transport receptor family. Aim number 2 is to test the hypothesis that this protein is a bona fide transporter, by identifying its cargo and showing that it mediates the translocation of this cargo across the nuclear pore. Aim number 1 experiments test the transporter for Ran (a small GTPase) binding, inhibition of the factors that influence the nucleotide state of Ran, and interactions with nuclear pore proteins. Experimental approaches for Aim number 2 include pulldown assays with immobilized transporter and cell lysates and in vitro transport assays in permeabilized cells. In the longer term, efforts will focus on mapping the targeting sequence(s) that sorts cargo for karyopherin X-mediated transport.